IFN-γ Expression Research Articles

Denfivontinib activates effector T-cells through NLRP3-inflammasome, yielding potent anticancer effects by combination with pembrolizumab.

Various combination therapies have been investigated to overcome the limitations of using immune checkpoint inhibitors. However, determining the optimal combination therapy remains challenging. To overcome the therapeutical limitation, we conducted a translational research to elucidate the mechanisms by which AXL inhibition enhances the anti-tumor effects when combined with anti-PD-1 antibody therapy. Herein, we demonstrated improved antitumor effects through combination treatment with denfivontinib and pembrolizumab which resulted in enhanced differentiation into effector CD4+ and CD8+ memory T cells, accompanied by an increase in IFN-γ expression in the YHIM-2004 xenograft model derived from patients with NSCLC. Concurrently, a reduction in the number of immunosuppressive M2 macrophages and myeloid-derived suppressor cells was observed. Mechanistically, denfivontinib potentiated the NOD-like receptor pathway, thereby facilitating the NLRP3 inflammasome formation. This leads to macrophage activation via the NF-kB signaling pathway activation. We have confirmed that the positive interaction between macrophages and T cells arises from the enhanced antigen-presenting machinery of activated macrophages. Furthermore, the observed tumor effects in AXL knock-out mice confirmed that AXL inhibition by denfivontinib enhances the anti-tumor effects, thus opening new avenues for therapeutic interventions aimed at overcoming limitations in immunotherapy. To demonstrate the extent to which our findings reflect clinical results, we analyzed bulk-RNA sequencing data from 21 NSCLC patients undergoing anti-PD-1 immunotherapy. The NLRP3 inflammasome score influenced enhanced immune responses in patient data undergoing anti-PD-1 immunotherapy, suggesting a role for NLRP3 inflammasome in activating immune responses during treatment.

Cholesterol promotes IFNG mRNA expression in CD4+ effector/memory cells by SGK1 activation.

IFNγ-secreting T cells are central for the maintenance of immune surveillance within the central nervous system (CNS). It was previously reported in healthy donors that the T-cell environment in the CNS induces distinct signatures related to cytotoxic capacity, CNS trafficking, tissue adaptation, and lipid homeostasis. These findings suggested that the CNS milieu consisting predominantly of lipids mediated the metabolic conditions leading to IFNγ-secreting brain CD4 T cells. Here, we demonstrate that the supplementation of CD4+CD45RO+CXCR3+ cells with cholesterol modulates their function and increases IFNG expression. The heightened IFNG expression was mediated by the activation of the serum/glucocorticoid-regulated kinase (SGK1). Inhibition of SGK1 by a specific enzymatic inhibitor significantly reduces the expression of IFNG Our results confirm the crucial role of lipids in maintaining T-cell homeostasis and demonstrate a putative role of environmental factors to induce effector responses in CD4+ effector/memory cells. These findings offer potential avenues for further research targeting lipid pathways to modulate inflammatory conditions.

Resveratrol alleviates Mono-2-ethylhexyl phthalate-induced mitophagy, ferroptosis, and immunological dysfunction in grass carp hepatocytes by regulating the Nrf2 pathway

Mono-2-ethylhexyl phthalate (MEHP) is the major biologically active metabolite of Di(2-ethylhexyl) phthalate (DEHP). This MEHP mono-ester metabolite can be transported through the bloodstream into tissues such as the liver, kidneys, fat, and testes and cause corresponding damage. Resveratrol (RSV) has anti-inflammatory, antioxidant, and detoxification characteristics. Our research examined whether RSV alleviates MEHP-induced grass carp hepatocyte (L8824 cell) injury and its relationship with the Nrf2 pathway, mitophagy, ferroptosis, and immune function. Therefore, we treated L8824 cells with 85 μM MEHP and/or 2 μM RSV. The findings indicated that exposing MEHP resulted in increased reactive oxygen species (ROS) content and decreased mitochondrial membrane potential in L8824 cells, which induced an up-regulation of the expression of mitophagy-related indicators (PINK1, Parkin, Beclin1, LC3B, and ATG5) and a down-regulation of P62. An up-regulation of the expression of the ferroptosis-related indicators TFR1 and COX-2, and GPX4 and FTH expression was down-regulated. In addition, there was a decrease in the expression of IL-2 and IFN-γ and an increase in the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6 after exposure to MEHP. RSV activates the Nrf2 pathway and effectively alleviates MEHP-induced mitophagy, ferroptosis, and immunologic dysfunction of L8824 cells.

Sesquiterpenoids from Inula britannica and their potential mechanism for immunomodulation

The immune system serves as a role of diseases, such as Parkinson's disease, and acute lung injury. An immunoregulatory activity-directed separation depended on phorbol 12-myristate 13-acetate (PMA) plus ionomycin (Ion)-mediated Jurkat leukemic T cells was used for studying chemical constituents from Inula britannica L. in depth. Five previously undescribed aromatic sesquiterpenoid dimers inulabritanoids J−N (1−5) and a previously undescribed germacrane-type sesquiterpenoid britanicafanin F (6) were afforded from I. britannica as well as eight known sesquiterpenoids (7–14). Their structures were elucidated through 1D and 2D NMR, HRMS, and ECD spectra along with quantum chemical calculations. Immunomodulatory effects of compounds 1–14 were assayed in PMA plus Ion-mediated Jurkat cells, and indicated that compounds 8, 9, and 13 displayed significantly inhibitory effects toward IL-2 and IFN-γ. Further investigation of mechanism of action revealed that compound 13 inhibited phosphorylations of p38, ERK, and JNK to suppress c-Jun and c-Fos expressions, resulting in blocking the nuclear translocation of AP-1 (a complex of c-Jun and c-Fos) to regulate mRNA expressions of IL-2 and IFN-γ. Molecular docking analysis demonstrated that compound 13 could enter into the cavity of p38, ERK, and JNK, and from hydrogen bond interactions with Gly33, Lys53 Ser154, and Asp168 for p38, Lys54, Glu71, Ser153, and Asp167 for ERK, and Met149 and Asn152 for JNK, which supported the abovementioned results. These findings suggested that sesquiterpenoids from the genus Inula served as immunomodulators for treating diseases involved in immune and inflammatory responses.

A novel pan-epitope based nanovaccine self-assembled with CpG enhances immune responses against flavivirus

BackgroundFlavivirus is a highly prevalent and outbreak-prone disease, affecting billions of individuals annually and posing substantial public health challenges. Vaccination is critical to reducing the global impact of flavivirus infections, making the development of a safe and effective vaccine a top priority. The self-assembled pan-epitope vaccine presents key advantages for improving immunogenicity and safety without relying on external vectors or adding immunomodulatory elements, both of which are essential for successful vaccine development.ResultsIn this study, the pan-epitope peptide TBT was combined with adjuvant CpG to form the TBT-CpG nanovaccine (TBT-CpG NaVs), which was found to be spherical, uniform in shape, and demonstrated strong serum stability. In vitro studies showed that the TBT-CpG NaVs were efficiently taken up and internalized by bone marrow-derived dendritic cells (BMDCs). Flow cytometry and transcriptomic analysis indicated that the antigens were effectively presented to antigen-presenting cells (APCs) via the MHC II pathway, which facilitated BMDCs maturation and promoted the release of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. In vivo studies confirmed that TBT-CpG NaVs enhanced antigen-specific IgG levels, significantly increased IFN-γ and IL-4 expression in spleen cells, and offered protective effects against Dengue virus (DENV) and Zika virus (ZIKV) infections. Safety evaluations revealed no hepatotoxicity and no significant organ damage in immunized mice.ConclusionThe self-assembled candidate nanovaccine TBT-CpG NaVs effectively activates BMDCs and triggers a targeted immune response, providing antiviral effects against DENV and ZIKV. This vaccine demonstrates good immunogenicity and safety, establishing a promising foundation and a new strategy for the development of safe and effective vaccines.Graphical

Dual therapy with corticosteroid ablates the beneficial effect of DP2 antagonism in chronic experimental asthma

Prostaglandin D2 (PGD2) signals via the DP1 and DP2 receptors. In Phase II trials, DP2 antagonism decreased airway inflammation and airway smooth muscle (ASM) area in moderate-to-severe asthma patients. However, in Phase III, DP2 antagonism failed to lower the rate of exacerbations, and DP2 as a target was shelved. Here, using a preclinical model of chronic experimental asthma, we demonstrate that rhinovirus-induced exacerbations increase PGD2 release, mucus production, transforming growth factor (TGF)-β1 and type-2 inflammation. DP2 antagonism or DP1 agonism ablates these phenotypes, increases epithelial EGF expression and decreases ASM area via increased IFN-γ. In contrast, dual DP1-DP2 antagonism or dual corticosteroid/DP2 antagonism, which attenuates endogenous PGD2, prevented ASM resolution. We demonstrate that DP2 antagonism resolves ASM remodelling via PGD2/DP1-mediated upregulation of IFN-γ expression, and that dual DP2 antagonism/corticosteroid therapy, as often occurred in the human trials, impairs the efficacy of DP2 antagonism by suppressing endogenous PGD2 and IFN-γ production.

Rapid Onset of Innate Response, Cytokine Signaling and Humoral Immunity in Inactivated LPAI-H9N2-Vaccinated Broilers

The development of effective and innovative vaccination strategies is urgently needed to better control the spread and transmission of the low-pathogenic avian influenza H9N2 subtype (LPAI-H9N2) in poultry. In addition, the enhancement of innate immunity by some of these innovative inactivated vaccines has not yet been investigated. Here, an experiment was conducted in commercial broiler chickens to compare the immune response to two different inactivated H9N2 vaccines. For this, Group 1 (G1) broilers were vaccinated with vaccine 1 [Nobilis® H9N2-P (pathogen-associated molecular patterns—PAMP) technology], broilers in G2 were vaccinated with vaccine 2 [an inactivated whole H9N2 virus (IWV) autogenous oil emulsion vaccine], while birds in G3 were not vaccinated. The study lasted 34 days. Innate immune parameters (phagocytic activity, nitric oxide, and lysozyme), cytokine signaling (IL-1β, IL-6, IL-8), humoral immunity using the hemagglutination inhibition (HI) test, and the gene expressions of IFN-γ and TLR-21 were assessed. The results showed a significant increase in innate immunity and modulatory cytokines at 24–48 h after the vaccination of G1 broilers, with a continuous increase until the end of the experiment. In addition, a significant increase in geometric mean HI titers was observed in G1 at 11 days post-vaccination (dpv), and a significant (p ˂ 0.05) upregulation of IFN-γ and TLR-21 was observed in the same group, G1, at 31 dpv compared to G2 and G3. Nobilis® H9N2-P may induce faster and stronger innate and active humoral immunity compared to another IWV, which may contribute to the protection of broilers against early H9N2 infections. However, challenge protection studies for several IWV vaccines, including PAMP-H9N2 against LPAI-H9N2, should be further evaluated in both specific pathogen-free (SPF) and commercial broilers.

Response-Adaptive Surgical Timing in neoadjuvant immunotherapy demonstrates enhanced pathologic treatment response in Head and Neck Squamous Cell Carcinoma.

We evaluated whether IDO-inhibitor BMS986205 (IDOi) + PD-1 inhibitor nivolumab enhanced T-cell activity and augmented immune-mediated antitumor responses in untreated, resectable HNSCC. We employed response-adaptive surgical timing to identify responders to immunotherapy and enhance their response. Patients with HNSCC were 3:1 randomized to receive nivolumab with or without BMS986205 PO daily (NCT03854032). In the combination arm, BMS986205 was initiated 7 days prior to nivolumab. Patients were stratified by HPV status. Response-adaptive surgical timing involved response assessment by radiographic criteria 4 weeks after nivolumab in both arms. Non-responders underwent surgical resection, while responders received 4 more weeks of randomized therapy before surgery. Biomarker analysis utilized pathologic treatment response (pTR) and RNA sequencing. Forty-two patients were enrolled, and the addition of IDOi to nivolumab did not result in greater rate of radiographic response (p=0.909). Treatment was well-tolerated with only 2 (5%) patients experiencing grade 3 immune-related adverse events. The addition of IDO-inhibitor augmented rates of pTR in patients with high baseline IDO RNA expression (p<0.05). Response-adaptive surgical timing demonstrated reliability in differentiating pathologic responders versus non-responders (p=0.009). A pretreatment NK cell signature, PD-L1 status, and IFN-g expression in the HPV- cohort correlated with response. HPV+ cohort found B-cell and CAF signatures predictive of response/non-response. Response-adaptive surgical timing enhanced treatment response. IDO-inhibitor BMS986205 augmented pTR in patients with high IDO1-expression in baseline samples, indicating a need for identifying and targeting resistant nodes to immunotherapy. HPV-status-dependent signatures predicting response to immunotherapy in HNSCC warrant further study.

Interleukin-2-mediated NF-κB-dependent mRNA splicing modulates interferon gamma protein production.

Interferon-gamma (IFNγ) is a pleiotropic cytokine produced by natural killer (NK) cells during the early infection response. IFNγ expression is tightly regulated to mount sterilizing immunity while preventing tissue pathology. Several post-transcriptional effectors dampen IFNγ expression through IFNG mRNA degradation. In this study, we identify mRNA splicing as a positive regulator of IFNγ production. While treatment with the combination of IL-12 and IL-2 causes synergistic induction of IFNG mRNA and protein, defying transcription-translation kinetics, we observe that NK cells treated with IL-12 alone transcribe IFNG with introns intact. When NK cells are treated with both IL-2 and IL-12, IFNG transcript is spliced to form mature mRNA with a concomitant increase in IFNγ protein. We find that IL-2-mediated intron splicing occurs independently of nascent transcription but relies upon NF-κB signaling. We propose that while IL-12 transcriptionally induces IFNG mRNA, IL-2 signaling stabilizes IFNG mRNA by splicing detained introns, allowing for rapid IFNγ protein production. This study uncovers a novel role for cytokine-induced splicing in regulating IFNγ through a mechanism potentially applicable to other inflammatory mediators.

Role of IFN-γ, IL-33, and IL-35 concentrations in tuberculous pleural effusion in diagnosis and analysis of the effectiveness of combined tests

This study examines the diagnostic utility of the combined interleukin-33 (IL-33), interferon-γ (IFN-γ), and interleukin-35 (IL-35) test in tuberculous pleural effusion. Forty patients with pleural effusion of unknown etiology admitted to the hospital between December 2020 and December 2023 were selected as the study group. The patients were further categorized into tuberculous (TB) (n = 20) and malignant (n = 20) groups on the basis of their relevant data, while sera from 20 healthy medical checkups were used as control group. Thoracentesis was used to collect pleural fluid, and after its collection, enzyme-linked immunosorbent assay was utilized to detect the concentration content of IL-35, IL-33, and IFN-γ in pleural fluid of patients with different types of diseases. Data processing and analysis were executed through SPSS23.0 software to plot the receiver operating characteristic curve and compare the area of the area under the line of each index. Adenosine deaminase level was statistically higher in TB group patients (59.91 U/L) than in malignant group (14.31 U/L) (P &lt; .05). Pleural fluid lactate dehydrogenase levels did not differ significantly between the 2 groups (P &gt; .05). T-SPOT test had a higher positive rate in the TB group (85%) and a lower positive rate in the malignant group (45%). The TB group exhibited significantly higher levels of IFN-γ expression than the malignant group (P &lt; .05). The TB group had higher levels of IL-33 and IL-35 compared to the malignant group, with a statistically significant difference (P &lt; .05). The TB group had a greater level of IL-33 (81.05 ± 10.11 pg/mL) than the cytology-positive group (39.37 ± 9.63 pg/mL), while the difference was not statistically significant (P &gt; .05). The level of IL-35 (70.11 ± 10.37 pg/mL) in the TB group was lower than the level of IL-35 (72.13 ± 9.58 pg/mL) in the cytology-positive group. The specificity of the series of combined tests reached 95.81%, which was statistically superior to the single-factor test (P &lt; .05). In the TB group, IFN-γ and IL-33, IFN-γ and IL-35, and IL-33 and IL-35 showed positive correlation. Combined determination of the concentration levels of IL-35, IL-33, and IFN-γ is of value in the diagnosis of tuberculous pleurisy.

Evening cortisol levels are prognostic for progression-free survival in a prospective pilot study of head and neck cancer patients.

Cortisol rhythm disruptions predict early mortality in renal, colorectal, lung, and metastatic breast cancer. In head and neck cancer (HNC), various cortisol indices are known to correlate with adverse psychological and biological (e.g., inflammatory) outcomes, but links to mortality have yet to be demonstrated. We hypothesize that the prognostic value of diurnal cortisol aberrations will hold in HNC. Prior work leads us to predict that flattened or elevated diurnal cortisol profiles will be associated with elevations of serum inflammatory and tumor-promoting cytokines in this population, and that these immune markers would themselves predict poor progression-free survival. We prospectively recruited a pilot sample of HNC patients (N=40) at a multidisciplinary HNC clinic. Most patients presented with late-stage oral/oropharyngeal cancer, were older than 50, male, and subsequently received combined-modality (surgery and/or radiotherapy with or without chemotherapy) treatment with curative intent. Saliva was collected twice daily for six days to assess diurnal slope, mean, waking, and evening cortisol levels. Serum was assayed for an exploratory panel of inflammatory and tumor-promoting cytokines. Two years post study-entry, disease progression and survivorship status were abstracted from medical records. Bivariate correlations, linear regressions, and Cox Proportional Hazards models tested hypotheses. Elevations of evening cortisol and diurnal mean levels were each associated with shorter progression-free survival (evening: Hazard Ratio [HR]=1.848, 95% Confidence Interval [CI]=1.057-3.230, p=.031; diurnal mean: HR=2.662, 95% CI=1.115-6.355, p=.027). Bivariate correlations revealed that higher levels of the serum inflammatory marker interferon (IFN)-γ were linked with elevated evening (r=.405, p=.014) and mean (r=.459, p=.004) cortisol. Higher expression of IFN-γ also predicted poorer progression-free survival (HR=4.671, 95% CI=1.409-15.484, p=.012). Elevated evening and diurnal mean cortisol were both prognostic; suggesting cortisol secretion is both dysregulated and elevated among patients who subsequently experienced accelerated disease progression. These exploratory data from 40 HNC patients mirror relationships between cortisol and survival identified among patients with numerous other tumor types. This pilot study highlights the need for research on effects of cortisol rhythm disruption among HNC patients. Future research in larger samples should also examine the role of inflammatory and tumor-promoting factors-both systemically and within the tumor microenvironment-as potential mediators of cortisol rhythm disruption.

Immunomodulatory effect of compound Xuelian capsule on experimental autoimmune myasthenia gravis in mice

IntroductionThis study aimed to explore the immunomodulatory effects of Xuelian capsules in experimental autoimmune myasthenia gravis (EAMG) mice. MethodsFemale C57BL/6 mice were immunized with AChR-α subunit R97–116 peptide to induce EAMG. Mice were randomized into five groups: EAMG model (n = 8), prednisone (n = 7), and low-, middle-, and high-dose Xuelian capsule groups (n = 7, 7, 8). Hematological parameters and NLR were assessed using an automatic hematology analyzer. Serum AchR-Ab, IFN-γ, IL-4, IL-10 levels were measured by ELISA. Thymus pathology was evaluated by HE staining. Spleen mRNA levels of TGF-β, IL-17, IL-6, IFN-γ were quantified by qPCR, and spleen cell populations by flow cytometry. ResultsXuelian capsules significantly reduced the ratio of NLR in peripheral blood, the concentration of AchR-Ab, IFN-γ, and IL-4 in serum, the expression of TGF-β, IL-17, IL-6, and IFN-γ in the spleen, and the proportion of CD4+/IL-4+ and CD4+/IFN-γ+ cells in the spleen, while increasing the concentration of IL-10 in the serum and the proportion of CD4+/Treg cells in the spleen. It also improved pathological changes in the thymus tissue of mice. DiscussionOne of the possible mechanisms of Xuelian capsules in the treatment of myasthenia gravis is to improve the pathological structure of the thymus of EAMG mice by inhibiting inflammatory factors and improving immunity, which then affects the occurrence and development of inflammation to achieve anti-inflammation, thus improving myasthenia gravis (MG).

Abstract 4132968: CCR5 modulation enhances M-MDSCs functionality in early atherosclerosis

Background: The role of immune suppressive microenvironment, promulgated by the presence of Myeloid Derived Suppressor Cells (MDSC) have been the major focus of research delineating the underlying mechanisms of cardiovascular diseases (CVD). CCR5, a chemokine receptor, has been associated with both immunosuppressive and inflammatory phenotypes, however, the possible role of CCR5 pertaining to MDSCs in the development of atherosclerosis has not been elucidated yet. Therefore, we investigated the therapeutic potential of CCR5 modulation in early atherosclerosis progression. Methods: CCR5 receptor-ligand expression was studied with flow cytometry, real time PCR and immunocytochemistry in 65 individuals (25 Healthy controls, 20 young hypercholesterolemic individuals, and 20 stable [Coronary Artery Disease] CAD patients). To induce atherosclerosis, mice were fed high fat diet (HFD) for 6 weeks following partial ligation of left carotid artery in C57BL6 mice. MDSCs phenotype and functionality were assessed using CCR5 inhibitor in vitro (10 -8 M) and in vivo (i.p 3ng/day for 15 days) . M-MDSCs phenotype switch, atherosclerotic lesion development and plaque phenotype was studied in vivo . Results: We observed CCR5 elevation on Monocytic-MDSCs in the early phase of atherosclerosis. M-MDSCs displayed an intermediate phenotype with expression of both pro and anti-inflammatory mediators in early atherosclerosis, a similar trend was observed in inflammatory cytokines stimulated M-MDSCs ( in vitro ). Notably, DAPTA treatment shifted phenotype of M-MDSCs to anti-inflammatory profile with elevated expression of IL-10 and reduced expression of IFNγ, IL4, and IL22. Further, CCR5 modulation expanded IL10+M-MDSC in vivo . Additionally, DAPTA reduced the migratory potential, reduced cholesterol uptake and improved the functionality (suppression of T cell) of M-MDSCs. Blocking CCR5 with DAPTA led to the formation of stable plaque in HFD-fed mice. Conclusion: Increased expression of CCR5 coupled with inflammatory cytokine secretion by regulatory cells lead to dysfunctional M-MDSCs in CVD risk factor population. Possibly this dysfunctionality contributes to the development and progression of CVD including atherosclerosis. Our findings show targeting of CCR5 with DAPTA reduces atherogenesis, increases plaque stabilization and corrects phenotypic alterations in immune suppressive M-MDSCs. The functionality and phenotype of M-MDSCs in atherosclerosis, may be ameliorated by targeting CCR5.

Koala ocular disease grades are defined by chlamydial load changes and increases in Th2 immune responses.

This study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis. A cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts. Analysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages. These findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies.

TMIC-50. IMPACT OF ZOTIRACICLIB ON GLIOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Zotiraciclib, an orally administered multi-kinase inhibitor, has shown anti-glioma effect via suppression of transcriptional process and mitochondrial function. Here, we investigate the impact of zotiraciclib on the glioma immune microenvironment. METHODS Mouse glioma was generated by intracranial injection of mouse syngeneic cells (IDH1-wildtype NPA cells or IDH1R132H-mutant NPAI cells) in C57BL/6 mice. Zotiraciclib was administered (30 mg/kg, intraperitoneal injection, twice per week) starting seven days after tumor implantation. NPA and NPAI tumors were dissected after 10 and 22 days’ treatment, respectively. Immune cells in the tumor tissue were isolated and examined using flow cytometry. Primary human CD8+ cells and CD14+ monocytes isolated from the blood of a healthy donor were used to further investigate the effect of zotiraciclib (15 nM and 50 nM) on human cytotoxic T cell activation and immunosuppressive M2 macrophage polarization, respectively, using flow cytometry. RESULTS No significant difference in the abundance of T cells, myeloid cells, NK cells, NKT cells, DCs, and B cells between zotiraciclib-treated and vehicle-treated groups was detected in neither NPA nor NPAI tumors. The treatment-induced change in the abundance of cytotoxic T cells, helper T cells, macrophages, monocytes, neutrophils, M-MDSCs, PMN-MDSCs, Tregs, and M2 macrophages wasn’t observed. Zotiraciclib reduced the expansion of human CD8+ cytotoxic T cells, but did not affect the expression of CD25, HLA-DR, TNFα and IFNγ after activation in vitro. The viability of human M2 macrophages during differentiation and polarization was not affected, while the expression of CD163 and CD206 was reduced by zotiraciclib. CONCLUSIONS The immunophenotyping performed for this study demonstrates that zotiraciclib does not alter the mouse glioma immune microenvironment. Complementary studies using human immune cells shows that zotiraciclib does not suppress the activation of cytotoxic T cells but reduces the polarization of immunosuppressive M2 macrophages, supporting combining zotiraciclib and immune stimulatory approach in glioma treatment.

IMMU-33. REPROGRAMMING OF MYELOID CELL METABOLISM VIA HEME OXYGENASE-1 INHIBITION POTENTIATES ANTI-TUMOR IMMUNITY IN A MURINE GLIOMA MODEL

Abstract Cancer cells exhibit a preference for aerobic glycolysis, a phenomenon known as the Warburg effect. Emerging evidence suggests that immune cells within the tumor microenvironment (TME) may employ a similar metabolic adaptation. Heme oxygenase-1 (HMOX-1), a metabolic regulatory gene, is elevated in myeloid cells of glioblastoma (GBM), yet its precise role in modulating immune suppression and energy utilization remains unclear. Our objective was to explore how targeting HMOX-1 activity in the GBM TME affects immune response and energy metabolism. We utilized syngeneic glioma models with C57BL/6J mice implanted with CT-2A cells, which were randomized into four treatment arms: intracranial (IC) injection of the HMOX-1 inhibitor zinc protoporphyrin (ZnPP), intraperitoneal (IP) injection of anti-PD-1, IC ZnPP + IP anti-PD-1, or IC saline (sham). For mechanistic studies, we developed a myeloid-specific knockdown model via adoptive cell transfer of myeloid-derived suppressor cells (MDSCs) transfected with Hmox-1 siRNA into CSF-1R myeloid-depleted mice. Immunophenotyping was conducted with flow cytometry. The Seahorse mitochondrial stress test was used for evaluation of energy dynamics. Downregulation of myeloid Hmox-1 expression and inhibition of HMOX-1 via ZnPP both led to heightened T cell activation, with increased IFN-γ expression in brain compared to untreated mice (P=0.039, 0.02, respectively). Notably, ZnPP treatment induced a compensatory elevation of PD-L1 in myeloid cells (P&amp;lt;0.0001). Both ZnPP monotherapy and anti-PD-1 + ZnPP combination therapy demonstrated enhanced median overall survival rates in comparison to sham and anti-PD-1 monotherapy groups, with the combined approach yielding the most significant survival advantage (P&amp;lt;0.0001). Metabolic analyses following Hmox-1 knockdown and ZnPP treatment revealed a shift away from cancer-like glycolytic shunting in MDSCs. Inhibiting the HMOX-1 pathway facilitated T cell activation and reversal of glycolysis-predominant metabolism in MDSCs, resulting in improved survival outcomes in a murine glioma model. Modulation of metabolic reprogramming in myeloid cells may potentiate anti-tumor immunity, especially in conjunction with checkpoint blockade.

Qingrexiaoji Recipe Regulates the Differentiation of M2 TAM via miR-29 in GC.

Gastric cancer, one of the most familiar adenocarcinomas of the gastrointestinal tract, ranks third in the world in cancer-related deaths. Traditional Chinese medicine can suppress the growth of tumors, and the underlying mechanism may be associated with the tumor microenvironment. Here, we investigated the anti-cancer effects of the Qingrexiaoji recipe on gastric cancer and the underlying molecular mechanism. An in vivo nude mouse model was established, and the expression of CD206, CD80, and M2 phenotype-related proteins (Arg-1, Fizz1) was obtained by flow cytometry and western blotting. The expressions of the M2 phenotype-related cytokines were examined by ELISA. Qingrexiaoji recipe inhibited gastric tumor growth and downregulated the expression of CD206, IFN-γ, IL-13, IL-4, and TNF-α in vivo. Qingrexiaoji recipe deceased M2 phenotypic polarization by upregulating microRNA (miR)-29a-3p level. Luciferase activity assays showed that HDAC4 is a potential target of miR-29a-3p. In cells co-transfected with HDAC4 siRNA and miR-29a-3p inhibitor and treated with IL-4 and Qingrexiaoji recipe, the miR-29a-3p inhibitorinduced increase of M2 phenotypic polarization was reversed. In summary, these results suggested that the Qingrexiaoji recipe regulated M2 macrophage polarization by regulating miR-29a-3p/HDAC4, providing a different and innovative treatment for gastric cancer.

Targeting METTL3 as a checkpoint to enhance T cells for tumour immunotherapy.

Immunotherapy has emerged as a crucial treatment modality for solid tumours, yet tumours often evade immune surveillance. There is an imperative to uncover novel immune regulators that can boost tumour immunogenicity and increase the efficacy of immune checkpoint blockade (ICB) therapy. Epigenetic regulators play critical roles in tumour microenvironment remodelling, and N6-methyladenosine (m6A) is known to be involved in tumourigenesis. However, the role of m6A in regulating T-cell function and enhancing anti-tumour immunity remains unexplored. Several cancer cell lines were treated with STM2457, an enzymatic inhibitor of RNA m6A methyltransferase METTL3, and explored the transcriptome changes with RNA sequencing (RNA-seq). We then utilised mouse melanoma (B16) and mouse colorectal adenocarcinoma (MC38) models to investigate the effects of METTL3 inhibition on immunotherapy, and analysed the dynamics of the tumour microenvironment via single-cell RNA-seq (scRNA-seq). Furthermore, in vitro and in vivo T-cell cytotoxicity killing assay and CRISPR Cas9-mediated m6A reader YTHDF1-3 knockout in B16 were performed to assess the role and the molecular mechanism of RNA m6A in tumour killing. Finally, the efficacy of METTL3 inhibition was also tested on human melanoma model (A375) and human T cells. We demonstrate that inhibiting METTL3 augments tumour immunogenicity and sustains T-cell function, thereby enhancing responsiveness to ICB therapy. Mechanistically, METTL3 inhibition triggers an interferon response within tumour cells, amplifying the anti-tumour immune response, along with deletion of the m6A reader protein YTHDF2 in tumours inhibiting major histocompatibility complex (MHC)-I degradation. Remarkably, these anti-tumour effects are reliant on the immune system. Specifically, METTL3 inhibition enhances interferon-gamma (IFNγ) and granzyme B (GzmB) expression, thereby strengthening T-cell killing ability, and concurrently dampening the expression of exhaustion-related genes. Targeting METTL3 enhances anti-tumour immunity by boosting T-cell cytotoxicity and reversing T-cell exhaustion. Our study positions METTL3 as an epigenetic checkpoint, highlighting the potential of targeting METTL3 to invigorate intrinsic anti-tumour defenses and overcome immune resistance. Targeting METTL3 augments tumour cell immunogenicity and sustains T-cell function. T cell with METTL3 inhibition can reverse T-cell exhaustion, and promote expression of IFNγ and GzmB, thereby enhancing cytotoxicity in anti-PD-1 therapy. YTHDF2 deletion in tumours prolong the lifespan of MHC-I mRNAs.

Comparison οf Immune Responses Through Multiparametric T-Cell Cytokine Expression Profile Between Children with Convalescent COVID-19 or Multisystem Inflammatory Syndrome.

The immunological pathways that cause Multisystem Inflammatory Syndrome after SARS-CoV-2 infection in children (MIS-C) remain under investigation. The aim of this study was to prospectively compare the T-cell cytokine expression profile in unvaccinated children with acute MIS-C (MISC_A) before immunosuppression, convalescent MIS-C (one month after syndrome onset, MISC_C), convalescent COVID-19 (one month after hospitalization), and in healthy, unvaccinated controls. The intracellular expression of IL-4, IL-2, IL-17, IFNγ, TNF-α and Granzyme B, and the post SARS-CoV-2-Spike antigenic mix stimulation of T-cell subsets was analyzed by 13-color flow cytometry. Twenty children with a median age (IQR) of 11.5 (7.25-14) years were included in the study. From the comparison of the flow cytometry analysis of the 14 markers of MISC_A with the other three groups (MISC_C, post-COVID-19 and controls), significant differences were identified as follows: 1. CD4+IL-17+/million CD3+: 293.0(256.4-870.9) vs. 50.7(8.4-140.5); p-value: 0.03, vs. 96.7(89.2-135.4); p-value: 0.03 and vs. 8.7(0.0-82.4); p-value: 0.03, respectively; 2. CD8+IL-17+/million CD3+: 335.2(225.8-429.9) vs. 78.0(31.9-128.9) vs. 84.1(0.0-204.6) vs. 33.2(0.0-114.6); p-value: 0.05, respectively; 3. CD8+IFNγ+/million CD3+: 162.2(91.6-273.4) vs. 41.5(0.0-77.4); p-value: 0.03 vs. 30.3(0.0-92.8); p-value: 0.08, respectively. In children presenting with MIS-C one month after COVID-19 infection, T cells were found to be polarized towards IL-17 and IFNγ production compared to those with uncomplicated convalescent COVID-19, a finding that could provide possible immunological biomarkers for MIS-C detection.

Effects of dietary fish meal replacement by Periplaneta americana meal on growth, metabolism, intestinal health and resistance against Aeromonas hydrophila of Micropterus salmoides

An 8-week culture experiment was conducted to investigate the effects of partial replacement of fish meal (FM) with Periplaneta americana meal (PAM) on growth performance, body composition, serum biochemical parameters, intestinal health and resistance against Aeromonas hydrophila of largemouth bass, Micropterus salmoides. A total of 450 healthy largemouth bass with an initial body weight (IBW) of 14.55±0.09 g were randomly divided into five groups. Five experimental diets were prepared by replacing 0 % (P0), 15 % (P15), 30 % (P30), 45 % (P45) and 60 % (P60) of the FM in the basal diet with PAM, respectively. The results showed that final body weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) were no significant difference in P15 and P30 compared with the control group (P > 0.05). Crude protein of whole fish body was significantly increased and crude lipid was significantly decreased in all PAM groups (P < 0.05). In the P30, the antioxidant enzyme activities were significantly higher than those in the control and other experimental groups, and malondialdehyde (MDA) was significantly decreased (P < 0.05). The activities of α-amylase and pepsin increased significantly in P15 and P30 and decreased significantly in P45 and P60 (P < 0.05). Lipase was significantly increased in all groups except P15 than in the control group (P < 0.05). There were no significant differences in villus height (VH), crypt depth (CD), and muscle thickness (MT) among all groups (P > 0.05), but villus width (VW) was significantly decreased in P45 and P60 (P < 0.05). The relative expression of IL-10 and TGF-β1 were first up-regulated and then down-regulated, while IL-8 was opposite (P < 0.05). The relative expression of Casp3, Casp9 and IFN-γ were significantly up-regulated in P45 and P60 (P < 0.05). After injecting A. hydrophila, all groups of largemouth bass experienced mortality, but cumulative mortality was lowest in the P30. In conclusion, PAM can partially replace FM in the diet of juvenile largemouth bass. The replacement of no more than 30 % is unlikely to adversely affect growth performance, and can increase the crude protein content of the whole fish body, and is beneficial to intestinal health.